bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020‒08‒02
37 papers selected by
Viktor Korolchuk
Newcastle University


  1. EMBO J. 2020 Jul 27. e105696
    Cinque L, De Leonibus C, Iavazzo M, Krahmer N, Intartaglia D, Salierno FG, De Cegli R, Di Malta C, Svelto M, Lanzara C, Maddaluno M, Wanderlingh LG, Huebner AK, Cesana M, Bonn F, Polishchuk E, Hübner CA, Conte I, Dikic I, Mann M, Ballabio A, Sacco F, Grumati P, Settembre C.
      Lysosomal degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is emerging as a critical regulator of cell homeostasis and function. The recent identification of ER-phagy receptors has shed light on the molecular mechanisms underlining this process. However, the signaling pathways regulating ER-phagy in response to cellular needs are still largely unknown. We found that the nutrient responsive transcription factors TFEB and TFE3-master regulators of lysosomal biogenesis and autophagy-control ER-phagy by inducing the expression of the ER-phagy receptor FAM134B. The TFEB/TFE3-FAM134B axis promotes ER-phagy activation upon prolonged starvation. In addition, this pathway is activated in chondrocytes by FGF signaling, a critical regulator of skeletal growth. FGF signaling induces JNK-dependent proteasomal degradation of the insulin receptor substrate 1 (IRS1), which in turn inhibits the PI3K-PKB/Akt-mTORC1 pathway and promotes TFEB/TFE3 nuclear translocation and enhances FAM134B transcription. Notably, FAM134B is required for protein secretion in chondrocytes, and cartilage growth and bone mineralization in medaka fish. This study identifies a new signaling pathway that allows ER-phagy to respond to both metabolic and developmental cues.
    Keywords:   TFEB ; ER-phagy; FGF signaling; Fam134B; IRS1/PI3K signaling
    DOI:  https://doi.org/10.15252/embj.2020105696
  2. Autophagy. 2020 Jul 27. 1-2
    Yin Z, Klionsky DJ.
      Selective autophagy is critical for the regulation of cellular homeostasis in organisms from yeast to humans. This process is a specific degradation pathway for a wide variety of substrates including unwanted cytosolic components, such as protein aggregates, damaged and/or superfluous organelles, and pathogens. However, it has been less clear as to whether a protein complex or substructure of an organelle can be targeted for removal by selective autophagy. One example of such a substrate is the nuclear pore complex (NPC), a large macromolecular assembly that is present throughout the nuclear envelope. Here, we highlight two recent studies that demonstrate for the first time that NPCs are targeted for vacuolar degradation through selective autophagy.ABBREVIATIONS: AIM: Atg8-interacting motif; NE: nuclear envelope; NPC: nuclear pore complex; Nup: nucleoporin; PMN/micronucleophagy: piecemeal microautophagy of the nucleus.
    Keywords:  Autophagy; NPC-phagy; Nup159; cargo receptor; nuclear pore complex; selective autophagy
    DOI:  https://doi.org/10.1080/15548627.2020.1798199
  3. Mol Cell. 2020 Jul 28. pii: S1097-2765(20)30510-4. [Epub ahead of print]
    Zhao D, Zou CX, Liu XM, Jiang ZD, Yu ZQ, Suo F, Du TY, Dong MQ, He W, Du LL.
      Autophagic degradation of the endoplasmic reticulum (ER-phagy) is triggered by ER stress in diverse organisms. However, molecular mechanisms governing ER stress-induced ER-phagy remain insufficiently understood. Here we report that ER stress-induced ER-phagy in the fission yeast Schizosaccharomyces pombe requires Epr1, a soluble Atg8-interacting ER-phagy receptor. Epr1 localizes to the ER through interacting with integral ER membrane proteins VAPs. Bridging an Atg8-VAP association is the main ER-phagy role of Epr1, as it can be bypassed by an artificial Atg8-VAP tether. VAPs contribute to ER-phagy not only by tethering Atg8 to the ER membrane, but also by maintaining the ER-plasma membrane contact. Epr1 is upregulated during ER stress by the unfolded protein response (UPR) regulator Ire1. Loss of Epr1 reduces survival against ER stress. Conversely, increasing Epr1 expression suppresses the ER-phagy defect and ER stress sensitivity of cells lacking Ire1. Our findings expand and deepen the molecular understanding of ER-phagy.
    Keywords:  ER stress; ER-phagy; ER-plasma membrane contact; UPR; autophagy; autophagy receptor
    DOI:  https://doi.org/10.1016/j.molcel.2020.07.019
  4. Ageing Res Rev. 2020 Jul 22. pii: S1568-1637(20)30264-6. [Epub ahead of print] 101129
    Ajoolabady A, Aslkhodapasandhokmabad H, Aghanejad A, Zhang Y, Ren J.
      Mitophagy serves as a cardinal regulator in the maintenance of mitochondrial integrity, function, and cardiovascular homeostasis, through the fine control and governance of cellular metabolism, ATP production, redox balance, and mitochondrial quality and quantity control. As a unique form of selective autophagy, mitophagy specifically recognizes and engulfs long-lived or damaged (depolarized) mitochondria through formation of the double-membraned intracellular organelles - mitophagosomes, ultimately resulting in lysosomal degradation. Levels of mitophagy are reported to be altered in pathological settings including cardiovascular diseases and biological ageing although the precise nature of mitophagy change in ageing and ageing-associated cardiovascular deterioration remains poorly defined. Ample clinical and experimental evidence has depicted a convincing tie between cardiovascular ageing and altered mitophagy. In particular, ageing perturbs multiple enigmatic various signal machineries governing mitophagy, mitochondrial quality, and mitochondrial function, contributing to ageing-elicited anomalies in the cardiovascular system. This review will update novel regulatory mechanisms of mitophagy especially in the perspective of advanced ageing, and discuss how mitophagy dysregulation may be linked to cardiovascular abnormalities in ageing. We hope to pave the way for development of new therapeutic strategies against the growing health and socieconomical issue of cardiovascular ageing through targeting mitophagy.
    Keywords:  Ageing; Cardiovascular Diseases; Mitochondria; Mitophagy
    DOI:  https://doi.org/10.1016/j.arr.2020.101129
  5. Cell Signal. 2020 Jul 27. pii: S0898-6568(20)30207-2. [Epub ahead of print] 109730
    Atwood DJ, Pokhrel D, Brown CN, Holditch SJ, Bachu DM, Thorburn A, Hopp K, Edelstein CL.
      Cardiac hypertrophy is common in autosomal dominant polycystic kidney disease (ADPKD) patients. We found increased heart weight in Pkd1RC/RC and Pkd2WS25/+ mouse models of ADPKD. As there is a link between increased heart weight and mammalian target of rapamycin (mTOR), the aim of the study was to determine mTOR complex 1 and 2 signaling proteins in the heart in the Pkd1RC/RC mouse model of PKD. In 70 day old Pkd1RC/RC hearts, on immunoblot analysis, there was a large increase in p-AMPKThr172, a known autophagy inducer, and an increase in p-AktSer473 and p-AktThr308, but no increase in other mTORC1/2 proteins (p-S6Ser240/244, p-mTORSer2448). In 150 day old Pkd1RC/RC hearts, there was an increase in mTORC1 (p-S6Ser240/244) and mTOR-related proteins (p-AktThr308, p-GSK3βSer9, p-AMPKThr172). As the mTOR pathway is the master regulator of autophagy, autophagy proteins were measured. There was an increase in p-Beclin-1 (BECN1), an autophagy regulator and activating molecule in Beclin-1-regulated autophagy (AMBRA1), a regulator of Beclin that play a role in autophagosome formation, an early stage of autophagy. There was a defect in the later stage of autophagy, the fusion of the autophagosome with the lysosome, known as autophagic flux, as evidenced by the lack of an increase in LC3-II, a marker of autophagosomes, with the lysosomal inhibitor bafilomycin, in both 70 day old and 150 day old hearts. To determine the role of autophagy in causing increased heart weight, Pkd1RC/RC were treated with 2-deoxyglucose (2-DG) or Tat-Beclin1 peptide, agents known to induce autophagy. 2-DG treatment from 150 to 350 days of age, a time period when increased heart weight developed, did not reduce the increased heart weight. Unexpectedly, Tat-Beclin 1 peptide treatment from 70 to 120 days of age resulted in increased heart weight. In summary, there is suppressed autophagic flux in the heart at an early age in Pkd1RC/RC mice. Increased mTOR signaling in older mice is associated suppressed autophagic flux. There was a large increase in p-AMPKThr172, a known autophagy inducer, in both young and old mice. 2-DG treatment did not impact increased heart weight and Tat-Beclin1 peptide increased heart weight.
    Keywords:  Autophagy; Autosomal dominant polycystic kidney disease; Cardiac hypertrophy; Heart; mTOR
    DOI:  https://doi.org/10.1016/j.cellsig.2020.109730
  6. Nat Metab. 2020 Jul 27.
    Orozco JM, Krawczyk PA, Scaria SM, Cangelosi AL, Chan SH, Kunchok T, Lewis CA, Sabatini DM.
      The mechanistic target of rapamycin complex 1 (mTORC1) kinase regulates cell growth by setting the balance between anabolic and catabolic processes. To be active, mTORC1 requires the environmental presence of amino acids and glucose. While a mechanistic understanding of amino acid sensing by mTORC1 is emerging, how glucose activates mTORC1 remains mysterious. Here, we used metabolically engineered human cells lacking the canonical energy sensor AMP-activated protein kinase to identify glucose-derived metabolites required to activate mTORC1 independent of energetic stress. We show that mTORC1 senses a metabolite downstream of the aldolase and upstream of the GAPDH-catalysed steps of glycolysis and pinpoint dihydroxyacetone phosphate (DHAP) as the key molecule. In cells expressing a triose kinase, the synthesis of DHAP from DHA is sufficient to activate mTORC1 even in the absence of glucose. DHAP is a precursor for lipid synthesis, a process under the control of mTORC1, which provides a potential rationale for the sensing of DHAP by mTORC1.
    DOI:  https://doi.org/10.1038/s42255-020-0250-5
  7. EMBO Rep. 2020 Jul 27. e202051175
    Zhang P, Edgar BA.
      The gastrointestinal tract undergoes homeostatic self-renewal to replace aged and damaged epithelial cells. This process, sustained by intestinal stem cells (ISCs), can operate accurately for many years but gradually declines with age. Although stem cell aging has been intensively explored, the mechanisms remain poorly understood. In this issue of EMBO Reports, Du et al report that alpha-lipoic acid (ALA) sustains an active endocytosis-autophagy network that effectively reverses age-dependent ISC hyperplasia in Drosophila (Du et al, 2020). This work suggests a new strategy for treating aging-associated gastrointestinal diseases.
    DOI:  https://doi.org/10.15252/embr.202051175
  8. EMBO J. 2020 Jul 27. e105965
    Fraiberg M, Elazar Z.
      The endoplasmic reticulum (ER) is a dynamic intracellular network responsible for folding and maturation of organellar and secreted proteins. Selective autophagy of ER (ER-phagy) is emerging as an essential process that maintains proteostasis in the ER and is regulated by growth conditions. In this issue, Cinque et al (2020) show that fibroblast growth factor 18 (FGF18) specifically activates ER-phagy through a TFEB/TFE-dependent transcriptional regulation of the ER-phagy receptor Fam134b, a process essential for bone ossification and skeletal development.
    DOI:  https://doi.org/10.15252/embj.2020105965
  9. FASEB J. 2020 Jul 28.
    Go YM, Zhang J, Fernandes J, Litwin C, Chen R, Wensel TG, Jones DP, Cai J, Chen Y.
      The retinal pigment epithelium (RPE) is a particularly vulnerable tissue to age-dependent degeneration. Over the life span, the RPE develops an expanded endo-lysosomal compartment to maintain the high efficiency of phagocytosis and degradation of photoreceptor outer segments (POS) necessary for photoreceptor survival. As the assembly and activation of the mechanistic target of rapamycin complex 1 (mTORC1) occur on the lysosome surface, increased lysosome mass with aging leads to higher mTORC1 activity. The functional consequences of hyperactive mTORC1 in the RPE are unclear. In the current study, we used integrated high-resolution metabolomic and genomic approaches to examine mice with RPE-specific deletion of the tuberous sclerosis 1 (Tsc1) gene which encodes an upstream suppressor of mTORC1. Our data show that RPE cells with constitutively high mTORC1 activity were reprogramed to be hyperactive in glucose and lipid metabolism. Lipolysis was suppressed, mitochondrial carnitine shuttle was inhibited, while genes involved in fatty acid (FA) biosynthesis were upregulated. The metabolic changes occurred prior to structural changes of RPE and retinal degeneration. These findings have revealed cellular events and intrinsic mechanisms that contribute to lipid accumulation in the RPE cells during aging and age-related degeneration.
    Keywords:  AMD; Mtor; aging; lipid; metabolism
    DOI:  https://doi.org/10.1096/fj.202000612R
  10. Proc Natl Acad Sci U S A. 2020 Jul 28. pii: 201920327. [Epub ahead of print]
    Choi WH, Yun Y, Park S, Jeon JH, Lee J, Lee JH, Yang SA, Kim NK, Jung CH, Kwon YT, Han D, Lim SM, Lee MJ.
      The 26S proteasome, a self-compartmentalized protease complex, plays a crucial role in protein quality control. Multiple levels of regulatory systems modulate proteasomal activity for substrate hydrolysis. However, the destruction mechanism of mammalian proteasomes is poorly understood. We found that inhibited proteasomes are sequestered into the insoluble aggresome via HDAC6- and dynein-mediated transport. These proteasomes colocalized with the autophagic receptor SQSTM1 and cleared through selective macroautophagy, linking aggresomal segregation to autophagic degradation. This proteaphagic pathway was counterbalanced with the recovery of proteasomal activity and was critical for reducing cellular proteasomal stress. Changes in associated proteins and polyubiquitylation on inhibited 26S proteasomes participated in the targeting mechanism to the aggresome and autophagosome. The STUB1 E3 Ub ligase specifically ubiquitylated purified human proteasomes in vitro, mainly via Lys63-linked chains. Genetic and chemical inhibition of STUB1 activity significantly impaired proteasome processing and reduced resistance to proteasomal stress. These data demonstrate that aggresomal sequestration is the crucial upstream event for proteasome quality control and overall protein homeostasis in mammals.
    Keywords:  STUB1; aggresome; proteaphagy; proteasome; ubiquitin
    DOI:  https://doi.org/10.1073/pnas.1920327117
  11. Autophagy. 2020 Jul 28.
    Wu X, Zheng Y, Liu M, Li Y, Ma S, Tang W, Yan W, Cao M, Zheng W, Jiang L, Wu J, Han F, Qin Z, Fang L, Hu W, Chen Z, Zhang X.
      Mitophagy, the elimination of damaged mitochondria through autophagy, promotes neuronal survival in cerebral ischemia. Previous studies found deficient mitophagy in ischemic neurons, but the mechanisms are still largely unknown. We determined that BNIP3L/NIX, a mitophagy receptor, was degraded by proteasomes, which led to mitophagy deficiency in both ischemic neurons and brains. BNIP3L exists as a monomer and homodimer in mammalian cells, but the effects of homodimer and monomer on mitophagy are unclear. Site-specific mutations in the transmembrane domain of BNIP3L (S195A and G203A) only formed the BNIP3L monomer and failed to induce mitophagy. Moreover, overexpression of wild-type BNIP3L, in contrast to the monomeric BNIP3L, rescued the mitophagy deficiency and protected against cerebral ischemic injury. The macroautophagy/autophagy inhibitor 3-MA and the proteasome inhibitor MG132 were used in cerebral ischemic brains to identify how BNIP3L was reduced. We found that MG132 blocked the loss of BNIP3L and subsequently promoted mitophagy in ischemic brains. In addition, the dimeric form of BNIP3L was more prone to be degraded than its monomeric form. Carfilzomib, a drug for multiple myeloma therapy that inhibits proteasomes, reversed the BNIP3L degradation and restored mitophagy in ischemic brains. This treatment protected against either acute or chronic ischemic brain injury. Remarkably, these effects of carfilzomib were abolished in bnip3l -/- mice. Taken together, the present study linked BNIP3L degradation by proteasomes with mitophagy deficiency in cerebral ischemia. We propose carfilzomib as a novel therapy to rescue ischemic brain injury by preventing BNIP3L degradation.
    Keywords:  BNIP3L/NIX; carfilzomib; cerebral ischemia; mitophagy; ubiquitin-proteasome pathway
    DOI:  https://doi.org/10.1080/15548627.2020.1802089
  12. Neuropathol Appl Neurobiol. 2020 Jul 28.
    Sato M, Ohta T, Morikawa Y, Konno A, Hirai H, Kurauchi Y, Hisatsune A, Katsuki H, Seki T.
      AIMS: Chaperone-mediated autophagy (CMA) is a pathway involved in the autophagy lysosome protein degradation system. CMA has attracted attention as a contributing factor to neurodegenerative diseases since it participates in the degradation of disease-causing proteins. We previously showed that CMA is generally impaired in cells expressing the proteins causing spinocerebellar ataxias (SCAs). Therefore, we investigated the effect of CMA impairment on motor function and the neural survival of cerebellar neurons using the micro RNA (miRNA)-mediated knockdown of lysosome-associated protein 2A (LAMP2A), a CMA-related protein.METHODS: We injected adeno-associated virus serotype 9 vectors, which express green fluorescent protein (GFP) and miRNA (negative control miRNA or LAMP2A miRNA) under neuron-specific synapsin I promoter, into cerebellar parenchyma of 4-week old ICR mice. Motor function of mice was evaluated by beam walking and footprint tests. Immunofluorescence experiments of cerebellar slices were conducted to evaluate histological changes of cerebella.
    RESULTS: GFP and miRNA were expressed in interneurons (satellite cells and basket cells) in molecular layers and granule cells in the cerebellar cortices, but not in cerebellar Purkinje cells. LAMP2A knockdown in cerebellar neurons triggered progressive motor impairment, prominent loss of cerebellar Purkinje cells, interneurons, granule cells at the late stage, and astrogliosis and microgliosis from the early stage.
    CONCLUSIONS: CMA impairment in cerebellar interneurons and granule cells triggers the progressive ataxic phenotype, gliosis, and the subsequent degeneration of cerebellar neurons, including Purkinje cells. Our present findings strongly suggest that CMA impairment is related to the pathogenesis of various SCAs.
    Keywords:  LAMP2A; cerebellum; chaperone-mediated autophagy; gliosis; motor dysfunction; neurodegeneration; spinocerebellar ataxia
    DOI:  https://doi.org/10.1111/nan.12649
  13. Nat Metab. 2020 Jul 27.
    Pinkosky SL, Scott JW, Desjardins EM, Smith BK, Day EA, Ford RJ, Langendorf CG, Ling NXY, Nero TL, Loh K, Galic S, Hoque A, Smiles WJ, Ngoei KRW, Parker MW, Yan Y, Melcher K, Kemp BE, Oakhill JS, Steinberg GR.
      Long-chain fatty acids (LCFAs) play important roles in cellular energy metabolism, acting as both an important energy source and signalling molecules1. LCFA-CoA esters promote their own oxidation by acting as allosteric inhibitors of acetyl-CoA carboxylase, which reduces the production of malonyl-CoA and relieves inhibition of carnitine palmitoyl-transferase 1, thereby promoting LCFA-CoA transport into the mitochondria for β-oxidation2-6. Here we report a new level of regulation wherein LCFA-CoA esters per se allosterically activate AMP-activated protein kinase (AMPK) β1-containing isoforms to increase fatty acid oxidation through phosphorylation of acetyl-CoA carboxylase. Activation of AMPK by LCFA-CoA esters requires the allosteric drug and metabolite site formed between the α-subunit kinase domain and the β-subunit. β1 subunit mutations that inhibit AMPK activation by the small-molecule activator A769662, which binds to the allosteric drug and metabolite site, also inhibit activation by LCFA-CoAs. Thus, LCFA-CoA metabolites act as direct endogenous AMPK β1-selective activators and promote LCFA oxidation.
    DOI:  https://doi.org/10.1038/s42255-020-0245-2
  14. EMBO J. 2020 Jul 28. e103009
    Fan SJ, Kroeger B, Marie PP, Bridges EM, Mason JD, McCormick K, Zois CE, Sheldon H, Khalid Alham N, Johnson E, Ellis M, Stefana MI, Mendes CC, Wainwright SM, Cunningham C, Hamdy FC, Morris JF, Harris AL, Wilson C, Goberdhan DC.
      Exosomes are secreted extracellular vesicles carrying diverse molecular cargos, which can modulate recipient cell behaviour. They are thought to derive from intraluminal vesicles formed in late endosomal multivesicular bodies (MVBs). An alternate exosome formation mechanism, which is conserved from fly to human, is described here, with exosomes carrying unique cargos, including the GTPase Rab11, generated in Rab11-positive recycling endosomal MVBs. Release of Rab11-positive exosomes from cancer cells is increased relative to late endosomal exosomes by reducing growth regulatory Akt/mechanistic Target of Rapamycin Complex 1 (mTORC1) signalling or depleting the key metabolic substrate glutamine, which diverts membrane flux through recycling endosomes. Vesicles produced under these conditions promote tumour cell proliferation and turnover and modulate blood vessel networks in xenograft mouse models in vivo. Their growth-promoting activity, which is also observed in vitro, is Rab11a-dependent, involves ERK-MAPK-signalling and is inhibited by antibodies against amphiregulin, an EGFR ligand concentrated on these vesicles. Therefore, glutamine depletion or mTORC1 inhibition stimulates release from Rab11a compartments of exosomes with pro-tumorigenic functions, which we propose promote stress-induced tumour adaptation.
    Keywords:  Rab11(a); exosome; extracellular vesicle; mechanistic Target of Rapamycin; multivesicular body
    DOI:  https://doi.org/10.15252/embj.2019103009
  15. Dev Cell. 2020 Jul 12. pii: S1534-5807(20)30541-4. [Epub ahead of print]
    Zhao YG, Zhang H.
      In eukaryotic cells, various membrane-bound organelles compartmentalize diverse cellular activities in a spatially and temporally controlled manner. Numerous membraneless organelles assembled via liquid-liquid phase separation (LLPS), known as condensates, also facilitate compartmentalization of cellular functions. Emerging evidence shows that these two organelle types interact in many biological processes. Membranes modulate the biogenesis and dynamics of phase-separated condensates by serving as assembly platforms or by forming direct contacts. Phase separation of membrane-associated proteins participates in various trafficking events, such as clustering of vesicles for temporally controlled fusion and storage, and transport of membraneless condensates on membrane-bound organelles. Phase separation also acts in cargo trafficking pathways by sorting and docking cargos for translocon-mediated transport across membranes, by shuttling cargos through the nuclear pore complex, and by triggering the formation of surrounding autophagosomes for delivery to lysosomes. The coordinated actions of membrane-bound and membraneless organelles ensure spatiotemporal control of various cellular functions.
    Keywords:  autophagy; condensates; membrane-bound organelles; phase separation; vesicle trafficking
    DOI:  https://doi.org/10.1016/j.devcel.2020.06.033
  16. Proc Natl Acad Sci U S A. 2020 Jul 31. pii: 202008021. [Epub ahead of print]
    Wei W, Ruvkun G.
      Mitochondrial fission and fusion are highly regulated by energy demand and physiological conditions to control the production, activity, and movement of these organelles. Mitochondria are arrayed in a periodic pattern in Caenorhabditis elegans muscle, but this pattern is disrupted by mutations in the mitochondrial fission component dynamin DRP-1. Here we show that the dramatically disorganized mitochondria caused by a mitochondrial fission-defective dynamin mutation is strongly suppressed to a more periodic pattern by a second mutation in lysosomal biogenesis or acidification. Vitamin B12 is normally imported from the bacterial diet via lysosomal degradation of B12-binding proteins and transport of vitamin B12 to the mitochondrion and cytoplasm. We show that the lysosomal dysfunction induced by gene inactivations of lysosomal biogenesis or acidification factors causes vitamin B12 deficiency. Growth of the C. elegans dynamin mutant on an Escherichia coli strain with low vitamin B12 also strongly suppressed the mitochondrial fission defect. Of the two C. elegans enzymes that require B12, gene inactivation of methionine synthase suppressed the mitochondrial fission defect of a dynamin mutation. We show that lysosomal dysfunction induced mitochondrial biogenesis, which is mediated by vitamin B12 deficiency and methionine restriction. S-adenosylmethionine, the methyl donor of many methylation reactions, including histones, is synthesized from methionine by S-adenosylmethionine synthase; inactivation of the sams-1 S-adenosylmethionine synthase also suppresses the drp-1 fission defect, suggesting that vitamin B12 regulates mitochondrial biogenesis and then affects mitochondrial fission via chromatin pathways.
    Keywords:  interorganelle communication; methionine restriction; mitochondrial dynamics; vacuolar V-ATPase; vitamin B12
    DOI:  https://doi.org/10.1073/pnas.2008021117
  17. Autophagy. 2020 Jul 27.
    Montenegro-Venegas C, Annamneedi A, Hoffmann-Conaway S, Gundelfinger ED, Garner CC.
      Maintaining the integrity and function of the presynaptic neurotransmitter release apparatus is a demanding process for a post-mitotic neuron; the mechanisms behind it are still unclear. BSN (bassoon), an active zone scaffolding protein, has been implicated in the control of presynaptic macroautophagy/autophagy, a process we recently showed depends on poly-ubiquitination of synaptic proteins. Moreover, loss of BSN was found to lead to smaller synaptic vesicle (SV) pools and younger pools of the SV protein SV2. Of note, the E3 ligase PRKN/parkin appears to be involved in BSN deficiency-related changes in autophagy levels, as shRNA-mediated knockdown of PRKN counteracts BSN-deficiency and rescues decreased SV protein levels as well as impaired SV recycling in primary cultured neurons. These data imply that BSN and PRKN act in concert to control presynaptic autophagy and maintain presynaptic proteostasis and SV turnover at the physiologically required levels.
    Keywords:  active zone; autophagosomes; neurodegeneration; presynapse; synaptic vesicle; ubiquitination
    DOI:  https://doi.org/10.1080/15548627.2020.1801259
  18. Aging Cell. 2020 Jul 30. e13189
    De Risi M, Torromino G, Tufano M, Moriceau S, Pignataro A, Rivagorda M, Carrano N, Middei S, Settembre C, Ammassari-Teule M, Gardoni F, Mele A, Oury F, De Leonibus E.
      Autophagy agonists have been proposed to slow down neurodegeneration. Spermidine, a polyamine that acts as an autophagy agonist, is currently under clinical trial for the treatment of age-related memory decline. How Spermidine and other autophagy agonists regulate memory and synaptic plasticity is under investigation. We set up a novel mouse model of mild cognitive impairment (MCI), in which middle-aged (12-month-old) mice exhibit impaired memory capacity, lysosomes engulfed with amyloid fibrils (β-amyloid and α-synuclein) and impaired task-induced GluA1 hippocampal post-translation modifications. Subchronic treatment with Spermidine as well as the autophagy agonist TAT-Beclin 1 rescued memory capacity and GluA1 post-translational modifications by favouring the autophagy/lysosomal-mediated degradation of amyloid fibrils. These findings provide new mechanistic evidence on the therapeutic relevance of autophagy enhancers which, by improving the degradation of misfolded proteins, slow down age-related memory decline.
    Keywords:  GluA1; Spermidine; ageing; alpha-synuclein; amyloid fibrils; autophagy; mild cognitive impairment
    DOI:  https://doi.org/10.1111/acel.13189
  19. Am J Physiol Cell Physiol. 2020 Jul 29.
    Abou Sawan S, Mazzulla M, Moore DR, Hodson N.
      Skeletal muscle is a highly plastic tissue capable of remodeling in response to a range of physiological stimuli including nutrients and exercise. Historically, the lysosome has been considered an essentially catabolic organelle contributing to autophagy, phagocytosis, and exo/endocytosis in skeletal muscle. However, recent evidence has emerged of several anabolic roles for the lysosome including the requirement for autophagy in skeletal muscle mass maintenance, the discovery of the lysosome as an intracellular signaling hub for mTORC1 activation, and the importance of TFEB/lysosomal biogenesis-related signaling in the regulation of mTORC1-mediated protein synthesis. We therefore propose that the lysosome is an understudied organelle with the potential to underpin the skeletal muscle adaptive response to anabolic stimuli. Within this review we describe the molecular regulation of lysosome biogenesis and detail the emerging anabolic roles of the lysosome in skeletal muscle with particular emphasis on how these roles may mediate adaptations to chronic resistance exercise. Furthermore, given the well-established role of amino acids to support muscle protein remodeling, we describe how dietary proteins 'labeled' with stable isotopes could provide a complimentary research tool to better understand how lysosomal biogenesis, autophagy regulation, and/or mTORC1-lysosomal repositioning can mediate the intracellular usage of dietary amino acids in response anabolic stimuli. Finally, we provide avenues for future research with the aim of elucidating how the regulation of this important organelle could mediate skeletal muscle anabolism.
    Keywords:  Lysosomal Biogenesis; Lysosome; Resistance Exercise; TFEB; mTORC1
    DOI:  https://doi.org/10.1152/ajpcell.00241.2020
  20. J Biol Chem. 2020 Jul 28. pii: jbc.RA119.010794. [Epub ahead of print]
    Luu AR, Wong H, Agrawal V, Wise N, Handyside B, Lo MJ, Pacheco G, Felix JB, Giaramita A, d'Azzo A, Vincelette J, Bullens S, Bunting S, Christianson TM, Hague C, Lebowitz JH, Yogalingam G.
      Mutations in the galactosidase β 1 (GLB1) gene cause lysosomal β-galactosidase (β-Gal) deficiency and clinical onset of the neurodegenerative lysosomal storage disease, GM1 gangliosidosis. β-Gal and neuraminidase 1 (NEU1) form a multi-enzyme complex in lysosomes along with the molecular chaperone, protective protein cathepsin A (PPCA). NEU1 is deficient in the neurodegenerative lysosomal storage disease sialidosis, and its targeting to and stability in lysosomes strictly depend on PPCA. In contrast, β-Gal only partially depends on PPCA, prompting us to investigate the role that β-Gal plays in the multienzyme complex. Here, we demonstrate that β-Gal negatively regulates NEU1 levels in lysosomes by competitively displacing this labile sialidase from PPCA. Chronic cellular uptake of purified recombinant human β-Gal (rhβ-Gal) or chronic lentiviral-mediated GLB1 over-expression in GM1 gangliosidosis patient fibroblasts coincides with profound secondary NEU1 deficiency. A regimen of intermittent enzyme replacement therapy (ERT) dosing with rhβ-Gal, followed by enzyme withdrawal, is sufficient to augment β-Gal activity levels in GM1 gangliosidosis patient fibroblasts without promoting NEU1 deficiency. In the absence of β-Gal, NEU1 levels are elevated in the GM1 gangliosidosis mouse brain, which are restored to normal levels following weekly intracerebroventricular (ICV) dosing with rhβ-Gal.  Collectively, our results highlight the need to carefully titrate the dose and dosing frequency of β-Gal augmentation therapy for GM1 gangliosidosis. They further suggest that intermittent ICV-ERT dosing with rhβ-Gal is a tunable approach that can safely augment β-Gal levels while maintaining NEU1 at physiological levels in the GM1 gangliosidosis brain.
    Keywords:  Beta-Galactosidase; Enzyme replacement therapy; GM1 gangliosidosis; Neuraminidase; PPCA; complex; gene therapy; genetic disease; neuraminidase; sialidase
    DOI:  https://doi.org/10.1074/jbc.RA119.010794
  21. Neuronal Signal. 2018 Dec;2(4): NS20180062
    Gao F, Zhang J.
      Mitochondria homeostasis is sustained by the mitochondrial quality control (MQC) system, which is crucial for cellular health, especially in the maintenance of functional mitochondria. A healthy mitochondria network is essential for life as it regulates cellular metabolism processes, particularly ATP production. Mitochondrial dynamics and mitophagy are two highly integrated processes in MQC system that determines whether damaged mitochondria will be repaired or degraded. Neurons are highly differentiated cells which demand high energy consumption. Therefore, compromised MQC processes and the accumulation of dysfunctional mitochondria may be the main cause of neuronal death and lead to neurodegeneration. Here, we focus on the inseparable relationship of mitochondria dynamics and mitophagy and how their dysfunction may lead to neurodegenerative diseases.
    Keywords:  mitochondria; neurodegeneration; quality control
    DOI:  https://doi.org/10.1042/NS20180062
  22. Front Cell Dev Biol. 2020 ;8 545
    Damiano V, Spessotto P, Vanin G, Perin T, Maestro R, Santarosa M.
      Autophagy is an intracellular catabolic process that is increasingly being recognized as a crucial factor in several human diseases including cancers. Mounting evidence suggests that autophagy allows tumor cells to overcome otherwise fatal stresses and to increase dissemination. Nevertheless, how autophagy controls these processes and in particular how it impinges on cell-cell adhesion is still poorly understood. Here, we investigate the role of autophagy in the turnover of the epithelial adhesion molecule E-cadherin in the context of breast cancer. We demonstrated in breast cancer cell lines that autophagy impinges on E-cadherin expression and in the configuration of adherens junctions. Besides, we showed that E-cadherin colocalizes with LC3B and SQSTM1/p62, two components of the autophagosome machinery. Pull down and immunoprecipitation analyses provided evidence that E-cadherin and SQSTM1/p62 physically interact. Moreover, the physical closeness of E-cadherin and SQSTM1/p62 was demonstrated by proximity ligation assays in breast cancer cell lines and primary tumors. Finally, we proved that the silencing of SQSTM1/p62 diminished the E-cadherin/LC3B colocalization, further supporting the role of SQSTM1/p62 in E-cadherin delivery to autophagosomes. These findings suggest that the activation of autophagy, reported in breast cancers with poor prognosis and in dormant breast cancer cells, may contribute to the control of tumor progression via downmodulation of E-cadherin protein levels.
    Keywords:  E-cadherin; SQSTM1; adherens junctions; autophagy; breast cancer
    DOI:  https://doi.org/10.3389/fcell.2020.00545
  23. Cell Calcium. 2020 Jul 17. pii: S0143-4160(20)30091-9. [Epub ahead of print]91 102249
    Pihán P, Hetz C.
      The endoplasmic reticulum (ER) is the source of lysosomal calcium. The finding that the protein TMBIM6 -a putative ER calcium channel and cell death regulator -promotes calcium transfer from the ER to lysosomes to induce autophagy uncovers a missing piece in the puzzle of inter-organelle communication.
    Keywords:  Autophagy; Calcium; ER stress; Lysosome; Protein misfolding; UPR
    DOI:  https://doi.org/10.1016/j.ceca.2020.102249
  24. Mol Biol Cell. 2020 Jul 29. mbcE20050291
    Dowdell AS, Cartwright IM, Goldberg MS, Kostelecky R, Ross T, Welch N, Glover LE, Colgan SP.
      Intestinal epithelial cells (IECs) exist in a metabolic state of low oxygen tension termed "physiologic hypoxia." An important factor in maintaining intestinal homeostasis is the transcription factor hypoxia-inducible factor (HIF), which is stabilized under hypoxic conditions and mediates IEC homeostatic responses to low oxygen tension. To identify HIF transcriptional targets in IEC, chromatin immunoprecipitation (ChIP) was performed in Caco-2 IECs using HIF-1α or HIF-2α specific antibodies. ChIP-enriched DNA was hybridized to a custom promoter microarray (termed ChIP-chip). This unbiased approach identified autophagy as a major HIF-1 targeted pathway in IEC. Binding of HIF-1 to the ATG9A promoter, the only transmembrane component within the autophagy pathway, was particularly enriched by exposure of IEC to hypoxia. Validation of this ChIP-chip revealed prominent induction of ATG9A, and luciferase promoter assays identified a functional hypoxia response element upstream of the TSS. Hypoxia-mediated induction of ATG9A was lost in cells lacking HIF-1. Strikingly, we found that lentiviral-mediated knockdown of ATG9A in IECs prevents epithelial barrier formation by >95% and results in significant mislocalization of multiple tight junction (TJ) proteins. Extensions of these findings showed that ATG9A knockdown cells have intrinsic abnormalities in the actin cytoskeleton, including mislocalization of the TJ binding protein vasodilator-stimulated phosphoprotein (VASP). These results implicate ATG9A as essential for multiple steps of epithelial TJ biogenesis and actin cytoskeletal regulation. Our findings have novel applicability for disorders that involve a compromised epithelial barrier and suggest that targeting ATG9A may be a rational strategy for future therapeutic intervention.
    DOI:  https://doi.org/10.1091/mbc.E20-05-0291
  25. Elife. 2020 Jul 28. pii: e56177. [Epub ahead of print]9
    Arriola Apelo SI, Lin A, Brinkman JA, Meyer E, Morrison M, Tomasiewicz JL, Pumper CP, Baar EL, Richardson NE, Alotaibi M, Lamming DW.
      Inhibition of mTOR (mechanistic Target Of Rapamycin) signaling by rapamycin promotes healthspan and longevity more strongly in females than males, perhaps because inhibition of hepatic mTORC2 (mTOR Complex 2) specifically reduces the lifespan of males. Here, we demonstrate using gonadectomy that the sex-specific impact of reduced hepatic mTORC2 is not reversed by depletion of sex hormones. Intriguingly, we find that ovariectomy uncouples lifespan from metabolic health, with ovariectomized females having improved survival despite paradoxically having increased adiposity and decreased control of blood glucose levels. Further, ovariectomy unexpectedly promotes midlife survival of female mice lacking hepatic mTORC2, significantly increasing the survival of those mice that do not develop cancer. In addition to identifying a sex hormone-dependent role for hepatic mTORC2 in female longevity, our results demonstrate that metabolic health is not inextricably linked to lifespan in mammals, and highlight the importance of evaluating healthspan in mammalian longevity studies.
    Keywords:  aging; genetics; genomics; healthspan; human biology; mTOR; mTORC2; medicine; mouse; ovariectomy; sex
    DOI:  https://doi.org/10.7554/eLife.56177
  26. Nature. 2020 Jul 29.
    Banik SM, Pedram K, Wisnovsky S, Ahn G, Riley NM, Bertozzi CR.
      The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein-for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins-the products of 40% of all protein-encoding genes7-are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified-but essential-component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.
    DOI:  https://doi.org/10.1038/s41586-020-2545-9
  27. Biochem Biophys Res Commun. 2020 Aug 27. pii: S0006-291X(20)31303-6. [Epub ahead of print]529(3): 596-602
    Tsuji-Tamura K, Sato M, Fujita M, Tamura M.
      Glycine, a non-essential amino acid, exerts concentration-dependent biphasic effects on angiogenesis. Low-doses of glycine promote angiogenesis, whereas high-doses cause anti-angiogenesis. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling participates in angiogenesis of both physiological development, and pathological events including tumor and inflammation. We assessed the role of PI3K/Akt/mTOR signaling in vascular development, and the interaction with glycine, using transgenic zebrafish Tg(fli1a:Myr-mCherry)ncv1 embryos expressing fluorescent proteins in vascular endothelial cells. Treatment with inhibitors of mTORC1 (rapamycin and everolimus), mTORC1/mTORC2 (KU0063794), PI3K (LY29400), and Akt (Akt inhibitor) decreased the development of intersegmental vessels (ISVs). These inhibitors cancelled the angiogenic effects of a low-dose of glycine, while acted synergistically with a high-dose of glycine in anti-angiogenesis. mTOR signaling regulates the gene expression of vascular endothelial growth factor (VEGF), a major angiogenic factor, and nitric oxide (NO) synthase (NOS), an enzyme for the synthesis of an angiogenic mediator NO. Expressions of VEGF and NOS were consistent with the vascular features induced by glycine and an mTOR inhibitor. Our results suggest that PI3K/Akt/mTOR signaling may interact with dose-dependent biphasic effects of exogenous glycine on in vivo angiogenesis. mTOR signaling is a key target for cancer therapy, thus, the combining mTOR inhibitors with glycine may be a potential approach for controlling angiogenesis.
    Keywords:  Akt; Angiogenesis; Glycine; PI3K; Zebrafish; mTOR
    DOI:  https://doi.org/10.1016/j.bbrc.2020.06.085
  28. Antioxidants (Basel). 2020 Jul 27. pii: E671. [Epub ahead of print]9(8):
    Lanzillotta C, Zuliani I, Vasavda C, Snyder SH, Paul BD, Perluigi M, Di Domenico FD, Barone E.
      Biliverdin reductase-A (BVR-A) impairment is associated with increased accumulation of oxidatively-damaged proteins along with the impairment of autophagy in the brain during neurodegenerative disorders. Reduced autophagy inhibits the clearance of misfolded proteins, which then form neurotoxic aggregates promoting neuronal death. The aim of our study was to clarify the role for BVR-A in the regulation of the mTOR/autophagy axis by evaluating age-associated changes (2, 6 and 11 months) in cerebral cortex samples collected from BVR-A knock-out (BVR-A-/-) and wild-type (WT) mice. Our results show that BVR-A deficiency leads to the accumulation of oxidatively-damaged proteins along with mTOR hyper-activation in the cortex. This process starts in juvenile mice and persists with aging. mTOR hyper-activation is associated with the impairment of autophagy as highlighted by reduced levels of Beclin-1, LC3, LC3II/I ratio, Atg5-Atg12 complex and Atg7 in the cortex of BVR-A-/- mice. Furthermore, we have identified the dysregulation of AMP-activated protein kinase (AMPK) as a critical event driving mTOR hyper-activation in the absence of BVR-A. Overall, our results suggest that BVR-A is a new player in the regulation of autophagy, which may be targeted to arrive at novel therapeutics for diseases involving impaired autophagy.
    Keywords:  AMPK; autophagy; biliverdin reductase; mTOR; neurodegeneration; oxidative stress
    DOI:  https://doi.org/10.3390/antiox9080671
  29. Nat Commun. 2020 Jul 30. 11(1): 3806
    Li ZL, Zhang HL, Huang Y, Huang JH, Sun P, Zhou NN, Chen YH, Mai J, Wang Y, Yu Y, Zhou LH, Li X, Yang D, Peng XD, Feng GK, Tang J, Zhu XF, Deng R.
      Most triple-negative breast cancer (TNBC) patients fail to respond to T cell-mediated immunotherapies. Unfortunately, the molecular determinants are still poorly understood. Breast cancer is the disease genetically linked to a deficiency in autophagy. Here, we show that autophagy defects in TNBC cells inhibit T cell-mediated tumour killing in vitro and in vivo. Mechanistically, we identify Tenascin-C as a candidate for autophagy deficiency-mediated immunosuppression, in which Tenascin-C is Lys63-ubiquitinated by Skp2, particularly at Lys942 and Lys1882, thus promoting its recognition by p62 and leading to its selective autophagic degradation. High Tenascin-C expression is associated with poor prognosis and inversely correlated with LC3B expression and CD8+ T cells in TNBC patients. More importantly, inhibition of Tenascin-C in autophagy-impaired TNBC cells sensitizes T cell-mediated tumour killing and improves antitumour effects of single anti-PD1/PDL1 therapy. Our results provide a potential strategy for targeting TNBC with the combination of Tenascin-C blockade and immune checkpoint inhibitors.
    DOI:  https://doi.org/10.1038/s41467-020-17395-y
  30. Front Immunol. 2020 ;11 1337
    Painter JD, Galle-Treger L, Akbari O.
      Autophagy is a cellular recycling system found in almost all types of eukaryotic organisms. The system is made up of a variety of proteins which function to deliver intracellular cargo to lysosomes for formation of autophagosomes in which the contents are degraded. The maintenance of cellular homeostasis is key in the survival and function of a variety of human cell populations. The interconnection between metabolism and autophagy is extensive, therefore it has a role in a variety of different cell functions. The disruption or dysfunction of autophagy in these cell types have been implicated in the development of a variety of inflammatory diseases including asthma. The role of autophagy in non-immune and immune cells both lead to the pathogenesis of lung inflammation. Autophagy in pulmonary non-immune cells leads to tissue remodeling which can develop into chronic asthma cases with long term effects. The role autophagy in the lymphoid and myeloid lineages in the pathology of asthma differ in their functions. Impaired autophagy in lymphoid populations have been shown, in general, to decrease inflammation in both asthma and inflammatory disease models. Many lymphoid cells rely on autophagy for effector function and maintained inflammation. In stark contrast, autophagy deficient antigen presenting cells have been shown to have an activated inflammasome. This is largely characterized by a TH17 response that is accompanied with a much worse prognosis including granulocyte mediated inflammation and steroid resistance. The cell specificity associated with changes in autophagic flux complicates its targeting for amelioration of asthmatic symptoms. Differing asthmatic phenotypes between TH2 and TH17 mediated disease may require different autophagic modulations. Therefore, treatments call for a more cell specific and personalized approach when looking at chronic asthma cases. Viral-induced lung inflammation, such as that caused by SARS-CoV-2, also may involve autophagic modulation leading to inflammation mediated by lung resident cells. In this review, we will be discussing the role of autophagy in non-immune cells, myeloid cells, and lymphoid cells for their implications into lung inflammation and asthma. Finally, we will discuss autophagy's role viral pathogenesis, immunometabolism, and asthma with insights into autophagic modulators for amelioration of lung inflammation.
    Keywords:  COVID-19; SARS-CoV-2; asthma; autophagy; immunometabolism; lung inflammation
    DOI:  https://doi.org/10.3389/fimmu.2020.01337
  31. PLoS One. 2020 ;15(7): e0233583
    Ormsby AR, Cox D, Daly J, Priest D, Hinde E, Hatters DM.
      Mutations that cause Huntington's Disease involve a polyglutamine (polyQ) sequence expansion beyond 35 repeats in exon 1 of Huntingtin. Intracellular inclusion bodies of mutant Huntingtin protein are a key feature of Huntington's disease brain pathology. We previously showed that in cell culture the formation of inclusions involved the assembly of disordered structures of mHtt exon 1 fragments (Httex1) and they were enriched with translational machinery when first formed. We hypothesized that nascent mutant Httex1 chains co-aggregate during translation by phase separation into liquid-like disordered aggregates and then convert to more rigid, amyloid structures. Here we further examined the mechanisms of inclusion assembly in a human epithelial kidney (AD293) cell culture model. We found mHttex1 did not appear to stall translation of its own nascent chain, or at best was marginal. We also found the inclusions appeared to recruit low levels of RNA but there was no difference in enrichment between early formed and mature inclusions. Proteins involved in translation or ribosome quality control were co-recruited to the inclusions (Ltn1 Rack1) compared to a protein not anticipated to be involved (NACAD), but there was no major specificity of enrichment in the early formed inclusions compared to mature inclusions. Furthermore, we observed co-aggregation with other proteins previously identified in inclusions, including Upf1 and chaperone-like proteins Sgta and Hspb1, which also suppressed aggregation at high co-expression levels. The newly formed inclusions also contained immobile mHttex1 molecules which points to the disordered aggregates being mechanically rigid prior to amyloid formation. Collectively our findings show little evidence that inclusion assembly arises by a discrete clustering of stalled nascent chains and associated quality control machinery. Instead, the machinery appear to be recruited continuously, or secondarily, to the nucleation of inclusion formation.
    DOI:  https://doi.org/10.1371/journal.pone.0233583
  32. Int J Mol Sci. 2020 Jul 27. pii: E5312. [Epub ahead of print]21(15):
    Shin JY, Muniyappan S, Tran NN, Park H, Lee SB, Lee BH.
      The 26S proteasome, a master player in proteolysis, is the most complex and meticulously contextured protease in eukaryotic cells. While capable of hosting thousands of discrete substrates due to the selective recognition of ubiquitin tags, this protease complex is also dynamically checked through diverse regulatory mechanisms. The proteasome's versatility ensures precise control over active proteolysis, yet prevents runaway or futile degradation of many essential cellular proteins. Among the multi-layered processes regulating the proteasome's proteolysis, deubiquitination reactions are prominent because they not only recycle ubiquitins, but also impose a critical checkpoint for substrate degradation on the proteasome. Of note, three distinct classes of deubiquitinating enzymes-USP14, RPN11, and UCH37-are associated with the 19S subunits of the human proteasome. Recent biochemical and structural studies suggest that these enzymes exert dynamic influence over proteasome output with limited redundancy, and at times act in opposition. Such distinct activities occur spatially on the proteasome, temporally through substrate processing, and differentially for ubiquitin topology. Therefore, deubiquitinating enzymes on the proteasome may fine-tune the degradation depending on various cellular contexts and for dynamic proteolysis outcomes. Given that the proteasome is among the most important drug targets, the biology of proteasome-associated deubiquitination should be further elucidated for its potential targeting in human diseases.
    Keywords:  RPN11; UCH37; USP14; deubiquitinating enzyme; deubiquitination; proteasome; proteolysis; ubiquitin
    DOI:  https://doi.org/10.3390/ijms21155312
  33. J Biol Chem. 2020 Jul 30. pii: jbc.RA120.013897. [Epub ahead of print]
    Nguyen N, Olivas TJ, Mires A, Jin J, Yu S, Luan L, Nag S, Kauffman KJ, Melia TJ.
      During autophagy, LC3 and GABARAP proteins become covalently attached to phosphatidylethanolamine (PE) on the growing autophagosome. This attachment is also reversible. Deconjugation (or delipidation) involves the proteolytic cleavage of an isopeptide bond between LC3 or GABARAP and the PE headgroup. This cleavage is carried about by the ATG4 family of proteases (ATG4A, B, C and D). Many studies have established that ATG4B is the most active of these proteases and is sufficient for autophagy progression in simple cells. Here we examine the second most active protease, ATG4A, to map out key regulatory motifs on the protein and to establish its activity in cells. We utilize fully in vitroreconstitution systems where we control the attachment of LC3/GABARAP members and discover a role for a carboxy-terminal (COOH-terminal) LC3-interacting region (LIR) on ATG4A in regulating its access to LC3/GABARAP. We then use a gene-edited cell line in which all four ATG4 proteases have been knocked out to establish that ATG4A is insufficient to support autophagy and is unable to support GABARAP proteins removal from the membrane. As a result, GABARAP proteins accumulate on membranes other than mature autophagosomes. These results suggest that to support efficient production and consumption of autophagosomes, additional factors are essential including possibly ATG4B itself, or one of its proteolytic products in the LC3 family.
    Keywords:  GABARAP Like-1; LC3 Interacting Region (LIR); MST4; autophagy; autophagy-related protein 4A (ATG4); cell biology; delipidation; liposome; membrane reconstitution; phosphorylation
    DOI:  https://doi.org/10.1074/jbc.RA120.013897
  34. PLoS One. 2020 ;15(7): e0236403
    Jung H, Seo SB.
      Autophagy, a self-degradative physiological process, is critical for homeostasis maintenance and energy source balancing in response to various stresses, including nutrient deprivation. It is a highly conserved catabolic process in eukaryotes and is indispensable for cell survival as it involves degradation of unessential or excessive components and their subsequent recycling as building blocks for the synthesis of necessary molecules. Although the dysregulation of autophagy has been reported to broadly contribute to various diseases, including cancers and neurodegenerative diseases, the molecular mechanisms underlying the epigenetic regulation of autophagy are poorly elucidated. Here, we report that the level of lysine demethylase 3B (KDM3B) increases in nutrient-deprived HCT116 cells, a colorectal carcinoma cell line, resulting in transcriptional activation of the autophagy-inducing genes. KDM3B was found to enhance the transcription by demethylating H3K9me2 on the promoter of these genes. Furthermore, we observed that the depletion of KDM3B inhibited the autophagic flux in HCT116 cells. Collectively, these data suggested the critical role of KDM3B in the regulation of autophagy-related genes via H3K9me2 demethylation and induction of autophagy in nutrient-starved HCT116 cells.
    DOI:  https://doi.org/10.1371/journal.pone.0236403
  35. Aging (Albany NY). 2020 Jul 26. 12
    Singh BK, Tripathi M, Sandireddy R, Tikno K, Zhou J, Yen PM.
      Although aging in the liver contributes to the development of chronic liver diseases such as NAFLD and insulin resistance, little is known about the molecular and metabolic details of aging in hepatic cells. To examine these issues, we used sequential oxidative stress with hydrogen peroxide to induce premature senescence in AML12 hepatic cells. The senescent cells exhibited molecular and metabolic signatures, increased SA-βGal and γH2A.X staining, and elevated senescence and pro-inflammatory gene expression that resembled livers from aged mice. Metabolic phenotyping showed fuel switching towards glycolysis and mitochondrial glutamine oxidation as well as impaired energy production. The senescent AML12 cells also had increased mTOR signaling and decreased autophagy which likely contributed to the fuel switching from β-oxidation that occurred in normal AML12 cells. Additionally, senescence-associated secretory phenotype (SASP) proteins from conditioned media of senescent cells sensitized normal AML12 cells to palmitate-induced toxicity, a known pathological effect of hepatic aging. In summary, we have generated senescent AML12 cells which displayed the molecular hallmarks of aging and also exhibited the aberrant metabolic phenotype, mitochondrial function, and cell signaling that occur in the aged liver.
    Keywords:  AML12 cells; aging; liver; metabolism; senescence
    DOI:  https://doi.org/10.18632/aging.103740
  36. Sci Rep. 2020 Jul 30. 10(1): 12827
    Dominguez-Meijide A, Vasili E, König A, Cima-Omori MS, Ibáñez de Opakua A, Leonov A, Ryazanov S, Zweckstetter M, Griesinger C, Outeiro TF.
      Parkinson's disease (PD) and Alzheimer's disease (AD) are common neurodegenerative disorders of the elderly and, therefore, affect a growing number of patients worldwide. Both diseases share, as a common hallmark, the accumulation of characteristic protein aggregates, known as Lewy bodies (LB) in PD, and neurofibrillary tangles in AD. LBs are primarily composed of misfolded α-synuclein (aSyn), and neurofibrillary tangles are primarily composed of tau protein. Importantly, upon pathological evaluation, most AD and PD/Lewy body dementia cases exhibit mixed pathology, with the co-occurrence of both LB and neurofibrillary tangles, among other protein inclusions. Recent studies suggest that both aSyn and tau pathology can spread and propagate through neuronal connections. Therefore, it is important to investigate the mechanisms underlying aggregation and propagation of these proteins for the development of novel therapeutic strategies. Here, we assessed the effects of different pharmacological interventions on the aggregation and internalization of tau and aSyn. We found that anle138b and fulvic acid decrease aSyn and tau aggregation, that epigallocatechin gallate decreases aSyn aggregation, and that dynasore reduces tau internalization. Establishing the effects of small molecules with different chemical properties on the aggregation and spreading of aSyn and tau will be important for the development of future therapeutic interventions.
    DOI:  https://doi.org/10.1038/s41598-020-69744-y
  37. Curr Neuropharmacol. 2020 Jul 28.
    Shi Q, Cheng Q, Chen C.
      Autophagy is a strictly regulated process which degrades and recycles long-lived or misfolded proteins and damaged organelles for the maintenance of energy and function homeostasis of cells. Insufficient oxygen and glucose supply caused by cerebral ischemia leads to higher ratio of AMP/ATP, which will activate AMPK pathway to initiate the process of autophagy. Accumulating evidence shows that autophagy participates in the pathogenesis of ischemic stroke as a doubleedge sword. However, the exact role of autophagy in the pathogenesis of ischemic stroke is controversial and yet to be elucidated. In this review, we expounded the autophagy pathway both in physiological condition and in ischemic stroke. We also focused on discussing the double-edge sword effect of autophagy in brain ischemia and its underlying mechanisms. In addition, we reviewed potential therapeutic strategies for ischemic stroke targeting autophagy pathway.
    Keywords:  autolysosome; autophagy; double-edge sword; ischemic stroke; pathogenesis; therapeutic strategies
    DOI:  https://doi.org/10.2174/1570159X18666200729101913